Comparative study of promoters for the production of polyhydroxyalkanoates in recombinant strains of Wautersia eutropha

Abstract

Recombinant strains of Wautersia eutropha expressing an artificial polyhydroxyalkanoate (PHA) biosynthesis operon under the control of different native promoters linked to polyhydroxybutyrate (PHB) (P(phb)), acetoin (P(acoE), P(acoD), and P(acoX)) or pyruvate (P(pdhE)) metabolism were constructed and tested. The promoters were representative either of the enterobacterial sigma70 (P(phb), P(acoE), and P(pdhE))- or sigma54 (P(acoD) and P(acoX))-dependent promoters. To obtain polymers consisting of C4-C12 monomer units, an artificial operon consisting of the PHA synthase gene from Pseudomonas sp. 61-3 (phaC1 (Ps)) tandemly linked to the W. eutropha genes encoding beta-ketothiolase (phbA (We)) and nicotinamide adenine dinucleotide phosphate dependent acetoacetyl-coenzyme A (CoA) reductase (phbB (We)) was constructed. All recombinant strains produced PHA, indicating that the PHA biosynthesis genes were expressed under the control of the different promoters. Cell growth and PHA synthesis on MS medium complemented with gluconate or octanoate, and different concentrations of acetoin (0, 0.15, and 0.3%) clearly differed among the recombinant strains. While the P(acoD) and P(acoX) promoters mediated only low PHA yields (<1%) in the presence of the inducer acetoin, the remaining promoters-independent of the addition of acetoin-resulted in the production of PHA polymers with high 3HB fractions (90-100 mol%) and with high 3HO contents (70-86 mol%) from gluconate and octanoate, respectively. Interestingly, on octanoate-MS medium with 0.15% acetoin, the P(acoE) promoter mediated the synthesis of PHA with a relatively high 3HB fraction (48 mol%). While PHAs with high 3HB contents were obtained, the overall PHA product yields were low (<10%); thus, their potential application for further commercial exploitation appears limited.

Evaluation of gene expression cassettes and production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) with a fine modulated monomer composition by using it in Cupriavidus necator

“…For instance, some popular and native promoters such as P lac , P lacUV5 , P tac , T7, P BAD , P phaC1 , P phaP , P acoE , P acoD , P acoX , and P pdhE , were investigated. Furthermore, P tac , which is known to be a constitutive strong promoter in Escherichia coli , functions well also in C. necator [12–16]. Additionally, highly stable vectors even under no antibiotic pressure have also been developed since various plasmid vectors are very unstable in C. necator H16.…”

A genome-wide approach for identification and characterisation of metabolite-inducible systems

“…This arrangement enables reporter protein synthesis in response to exogenous supplementation with the proposed ligand to be measured by fluorescence output. The Escherichia coli L-arabinose-, and the C. necator acetoin-inducible systems have been tested previously 24,25 and were included for comparative purpose (Fig. 2b).…”

- Synthetic Routes to Methylerythritol Phosphate Pathway Intermediates and Downstream Isoprenoids

“…coli consensus RBS sequence (pCM271THC) or pBBR1 origin/calculated RBS sequence (pBADTcalRBSHC) combinations did not dramatically reduce combined hydrocarbon titers, but produced a more balanced pentadecane:heptadecene ratio, we constructed plasmid pCM271TcalRBSHC to evaluate the hydrocarbon titer of the mutant pCM271 origin/calculated RBS sequence combination. Surprisingly, pCM271TcalRBSHC achieved a 6-fold improvement in combined hydrocarbon titer (~6 mg/L, Figure 3 the highest titer construct using previously established R. eutropha expression system components [7]. Furthermore, pCM271TcalRBSHC achieved a 100-fold improvement over the lowest production plasmids, pCMTHC and pYIUV5THC ( Figure 4).…”

“…Although suicide vectors have been used to generate inframe deletions and point mutations in R. eutropha [5], and previously reported broad-host range expression systems [6] may be transferable to R. eutropha, to date, the only established inducible expression system for R. eutropha has been a pBBR1-derived vector with a P BAD promoter [7]. Here, we have initiated the development of a synthetic biology toolbox to enable complex metabolic engineering applications in R. eutropha H16.…”